Cellular signaling is crucial in several different developmental pathways and adult homeostatic processes. For example, in cellular signaling, tight regulation of Hedgehog (Hh) signaling is essential in building and maintaining functional systems in the body. Hh signaling is implicated in many human diseases, notably several neurodegenerative diseases and a variety of cancers including basal cell carcinoma, medulloblastoma, rhabdomyosarcoma, pancreatic cancer, prostate cancer, and lung cancer.
The identification of mutations in Hh pathway components has stimulated the identification of over a dozen Hh agonists and antagonists that fall into chemically distinct classes. Most of these compounds target the protein Smoothened (Smo). In the mammalian signaling pathway, the binding of a Hh ligand to its receptor Patched 1 (Ptch1), relieves Ptch1-mediated inhibition of Smo, a second trans-membrane protein. The activation of Smo then triggers an intracellular signal transduction cascade that culminates in Gli-dependent (glioma-associated oncogene-dependent) transcriptional activities. Hence, a mechanistic understanding of Smo and how it interacts with potential signaling modulators is important for the development of effective drugs and therapies for Hh-related diseases. Current drug screening methods for Hh signaling pathway agonists and antagonists use downstream transcriptional activity as readout, which is indirect and does not distinguish between different Smo translocation mechanisms. In addition, previous methods have limited information depth, specificity, and statistical robustness Thus, there remains a need for additional methods, including high throughput methods, for screening modulators of Smo, Hh and other cellular signaling pathways.